Cells receive various stimuli from the outside and react in response to these stimuli. Examples of such stimuli include binding of cytokine or hormone to a receptor, viral infection, physical contact with other cells, thermal stimuli such as high or low temperature, pH, drug stimulus, stimulus from a substance such as nutrients and amino acids in the environment surrounding the cells and so forth. In many cases, when a cell reacts in response to such a stimulus, the stimulus is transmitted to the nucleus as a signal, and as a result, expression of a certain gene is regulated. As in the case of lipid-soluble hormones, some proteins pass through a cell membrane and nuclear membrane and directly bind to a receptor in the nucleus to regulate transcription. However, many signals are first captured on the cell membrane, and then a stimulus is passed onto signal transducing molecules and transmitted from the cytoplasm to the nucleus by the signal transducing molecules. Transport of some of these transducing molecules from the cytoplasm into the nucleus is known to be essential to the signal transduction. Several signal transducing molecules, which pass through a nuclear membrane, such as STAT1 (Science, 277: p.1630–1635 (1997)) and NFκB (Annu. Rev. Immunol., 16: p.225–260 (1998)), have been identified to date, but it is considered that there are still many unknown molecules.
A large number of proteins that are simply transported from the cytoplasm into the nucleus have been found by various research methods. Proteins constituting the nucleus or so-called nucleoproteins identified to date are synthesized as a peptide from mRNA in the cytoplasm and then transported into the nucleus. These proteins can also be regarded as proteins that are transported into the nucleus. Examples of the nucleus constituting proteins include nucleohistone, nucleoprotamine, lamin and so forth. However, many of these are not considered directly involved in the signal transduction.
As a method for searching for and identifying, not proteins that are constantly localized in the nucleus as described above, but proteins that are transported to the nucleus, there is an example in which a gene coding for a GFP protein, which emits self-fluorescence, was ligated with cDNA to express a fusion protein, so that a protein having a nuclear transport signal encoded by the cDNA portion was isolated by using fluorescence emitted by the GFP as a marker (The Journal of Cell Biology, 146, 29–43, 1999). However, with this method, proteins having a nuclear transport signal are only identified, and cDNA of proteins such as so-called signal transducing molecules, which are transported from the cytoplasm into the nucleus in response to a certain stimulus, cannot be isolated.
Meanwhile, TNF is released mainly from immune system cells upon inflammation and induces inflammation as a biophylactic reaction. However, when this action becomes excessive, various diseases are developed. For example, when hepatitis, which is caused by viral infection, is developed into chronic state, chronic production of TNF induces fibrosis of the liver, and progression of this condition causes cirrhosis. It is also known that inflammation is relieved by TNF antibodies in many diseases such as hepatitis, enteritis, pneumonia, nephritis and arthritis. In fact, commercially available anti-TNF antibodies show remarkable effects as therapeutic agents.
Furthermore, TNF is involved in the biophylactic reactions and plays an important role in activation of immunocompetent cells, repair of organs and elimination of tumors. In treatment of diseases associated with TNF, it is considered to be effective that TNF actions should not be totally suppressed, but that only a harmful part of the TNF actions should be suppressed or only a useful part should be enhanced.
TNF actions vary depending on the kind of the cell or the state of the cell, and TNF exhibits various actions. For example, in a liver cell, TNF binds to a TNF receptor and induces both a signal inducing apoptosis (death signal) and a signal inducing expression of various genes (gene expression inducing signal) via a transcription factor such as NFκB. The former signal is not accompanied by induction of gene expression. The latter signal induces gene expression, and a part of proteins produced thereby has an action for canceling the death signal (anti-apoptosis action). Usually, these two types of signals are balanced, and a normal cell exposed to TNF does not undergo apoptosis. When the anti-apoptosis action is inhibited due to some cause, a cell undergoes apoptosis. For example, fulminant hepatitis is a disease wherein a wide range of liver cells rapidly undergo apoptosis. Its mortality is extremely high, and no good therapy is available. It is considered that this is resulted from failure of the anti-apoptosis mechanism of the liver cells against TNF due to some cause and induction of only apoptosis signals by TNF.
Thus, various actions of TNF are considered to be attributable to actions of various transcription factors that transmit TNF signals.
A gene expression promoting signal induced by TNF induces gene expressions by a mechanism in which a certain protein receiving the signal from a TNF receptor is transported from the cytoplasm into the nucleus to form a certain complex and bind to a transcription regulating region so that the gene expression should be induced. The expressed genes show various aspects depending on the kind of the cell and influences of other signals that simultaneously exist. It is considered that each protein which is transported into the nucleus plays a different role and is involved in a different gene expression.
As the nuclear transport molecule that transmits TNF signals, NFκB is known. NFκB is a nuclear transport protein that leads presence of, not only TNF, but also many cytokines, growth factors and stress stimulus, to gene expressions. Considering diversity of the TNF actions, it seems impossible that NFκB should be the only transcription factor of TNF. Therefore, it is expected that there exist unknown proteins that are transported into the nucleus in response to TNF stimulus.
Disclosure of the Invention
An object of the present invention is to provide a method for searching for a gene coding for a protein that is specifically transported into a nucleus in response to a certain stimulus applied to a cell. Further, another object of the present invention is to provide a method for screening a therapeutic agent for a diseases caused by TNF stimulus by utilizing a protein which is transported from cytoplasm of a eukaryotic cell into a nucleus through a nuclear pore or a gene coding for the same.
The inventors of the present invention assiduously studied in order to achieve the above objects. As a result, they found that an object gene could be retrieved by cloning each gene in many gene groups into an expression vector so that its gene product should be expressed as a fusion protein with a protein emitting self-fluorescence to construct a gene expression library, introducing this gene expression library into two groups of cells so that the fusion proteins should be expressed in the cells, stimulating only one cell group by a drug or the like, isolating cells in which the fusion proteins were localized in the nuclei or the nuclei thereof from the cells of the two groups, respectively, and then comparing them. Thus, they accomplished the present invention.
That is, the following are provided by the present invention.    (1) A method for searching for a gene coding for a protein which is transported from cytoplasm into a nucleus through a nuclear pore in a eukaryotic cell, comprising the steps of:    (a) introducing a library of genes coding for fusion proteins of a marker protein whose localization in a cell can be detected and a part or whole of a search object protein into eukaryotic cells to allow expression of the genes in the transgenic cells so that the fusion proteins should be produced;    (b) dividing the transgenic cells into two groups and stimulating only cells of one group so that a nuclear transport protein should be transported into nuclei;    (c) detecting localization of the fusion proteins in cells by using the marker protein in each of the stimulated cell group and the unstimulated cell group and isolating cells in which the fusion proteins are localized in the nuclei or their nuclei; and    (d) recovering genes coding for a fusion protein from the isolated cells or their nuclei, and comparing genes recovered from the stimulated cell group and genes recovered from the unstimulated cell group.    (2) The method according to (1), which comprises, after the step (d), a step of identifying a gene recovered only from the stimulated cell group or a gene recovered only from the unstimulated cell group.    (3) The method according to (1) or (2), wherein, in the step (c), operations of detecting localization of the fusion proteins in a cell based on the marker protein and isolating cells or nuclei in which localization of the fusion proteins is observed in the nuclei are repeated twice or more for the isolated cells or nuclei.    (4) The method according to any one of (1) to (3), wherein the marker protein is a protein emitting self-fluorescence, a protein catalyzing color development reaction, an antigen peptide that can be detected with an antibody or a peptide that can bind to another substance.    (5) The method according to (4), wherein the protein emitting self-fluorescence is a green fluorescent protein.    (6) The method according to any one of (1) to (5), wherein, in the step (b), the transport of the nuclear transport protein into the nuclei is stimulated by means selected from the group consisting of a chemical substance, a physiologically active substance, environmental change and contact with other cells.    (7) The method according to any one of (1) to (6), wherein the cells or their nuclei are isolated by cell sorting, flow cytometry, microdissection or microscopic observation.    (8) The method according to any one of (1) to (7), wherein, in the step (c), permeability of the cell membranes is increased so that the fusion proteins in the cytoplasm should be flown out of the cells prior to the isolation of the cells.    (9) The method according to any one of (1) to (8), wherein, in the step (c), the cell membranes of the cells are disrupted and then the nuclei are isolated.    (10) The method according to any one of (1) to (9), wherein, in the step (d), the genes recovered from the stimulated cell group and the genes recovered from the unstimulated cell group are compared based on sequence analysis of full length or a part of a region coding for a search object protein for the genes.    (11) The method according to any one of (1) to (10), wherein, in the step (a), one or more genes coding for a fusion protein autonomously transporting into the nucleus with no stimulus are eliminated from the gene library beforehand.    (12) The method according to any one of (1) to (11), wherein, in the step (a), one or more genes coding for a fusion protein with an extranuclear protein, which is never transported into the nucleus, are eliminated from the gene library beforehand.    (13) The method according to any one of (1) to (12), wherein, in the step (a), the search object protein is a protein which regulates gene transcription.    (14) The method according to any one of (1) to (13), wherein, in the step (a), the fusion proteins further include a nuclear export signal.    (15) A method for screening a therapeutic agent for a disease caused by TNF stimulus, comprising the steps of:    (a) preparing eukaryotic cells expressing a protein which is transported from cytoplasm into a nucleus through a nuclear pore in response to TNF stimulus;    (b) allowing TNF and a test substance to act on the eukaryotic cells;    (c) detecting a protein transported into the nucleus; and    (d) selecting a test substance that accelerates or inhibits the nuclear transport of the protein when the test substance is allowed to act on the cells in comparison to a case where the test substance is not allowed to act on the cells.    (16) The method according to (15), wherein the eukaryotic cells express the protein which is transported into a nucleus because of introduction of a gene coding for the protein.    (17) The method according to (16), wherein the protein which is transported into a nucleus is a fusion protein with a marker protein whose localization in a cell can be detected.    (18) The method according to any one of (15) to (17), wherein the eukaryotic cells are transformant cells transformed with a foreign gene other than the gene coding for the protein, and an expression product of the foreign gene is the test substance.    (19) The method according to (18), wherein the eukaryotic cells are transformants introduced with a gene library.    (20) The method according to any one of (15) to (19), wherein the protein which is transported into a nucleus is a product of a gene selected from human genes of UniGene numbers Hs.12303, Hs.183180, Hs.198246, Hs.83849, Hs.76722, Hs.74034, Hs.129959, Hs.24301, Hs.24756, Hs.161137, Hs.348609 and Hs.24608.
Hereafter, the present invention will be explained in detail.
<1> Method for Screening Nuclear Transport Protein of the Present Invention
The first aspect of the present invention relates to a method for searching for a gene coding for a protein transported from cytoplasm of a eukaryotic cell into a nucleus through a nuclear pore. In the present invention, when transport of a protein from cytoplasm of a eukaryotic cell into a nucleus through a nuclear pore is referred to as “nuclear transport”. The protein which is transported into the nucleus is referred to as a “nuclear transport protein”. In the present invention, specifically, the nuclear transport protein is a protein that is transported into a nucleus when a cell is stimulated by a certain stimulus.
The method of the present invention comprises the following steps:    (a) introducing a library of genes coding for fusion proteins of a marker protein whose localization in a cell can be detected and a part or whole of a search object protein into eukaryotic cells to allow expression of the genes in transgenic cells so that the fusion proteins should be produced;    (b) dividing the transgenic cells into two groups and stimulating only cells of one group so that a nuclear transport protein should be transported into nuclei;    (c) detecting localization of the fusion proteins in cells by using the marker protein in each of the stimulated cell group and the unstimulated cell group and isolating cells in which the fusion proteins are localized in the nucleus or their nuclei; and    (d) recovering genes coding for a fusion protein from the isolated cells or their nuclei, and comparing genes recovered from the stimulated cell group and genes recovered from the unstimulated cell group.
Hereafter, the method of the present invention will be explained step by step.
(1) Step (a)
First, a gene library of genes coding for fusion proteins of a marker protein whose localization in a cell can be detected and a part or whole of a search object protein is constructed. The marker protein can be used to detect localization of the fusion proteins in a cell by detecting the marker protein itself, and is not particularly limited so long as it dose not substantially inhibit nuclear transport of the fusion proteins. As for the detection of the localization in a cell, the localization can be preferably visualized while substantially maintaining the cellular or nuclear structure. The marker protein may be one that can be detected directly or indirectly by a visual or electrical method or the like. Specific examples of the marker protein include a protein emitting self-fluorescence, protein catalyzing a color development reaction, antigen peptide that can be detected by an antibody or peptide that can specifically bind to anther substance.
As the protein emitting self-fluorescence, there can be mentioned, for example, a green fluorescent protein (GFP). The self-fluorescence used herein refers to light emitted by absorbing energy from outside such as excitation light, and its fluorescence wavelength is not particularly limited so long as it can be detected. Phosphorescence is also included. GFP is a protein recently found in Aequorea (Proc. Natl. Acad. Sci. USA, 91: 12501–12504 (1994)), and does not require a cofactor such as a metal ion for light emission unlike chlorophyll or the like. Therefore, GFP is suitable to find localization in a cell. Nuclei containing GFP can be isolated, for example, by sorting with a flow cytometer. Further, the position of nuclei can be confirmed by staining using Hoechst 33342 (Molecular Probes, Inc.) and the amount of GFP in the recognized nuclei can be quantified by using an ArrayScan system (Cellomics).
As the protein catalyzing a color development reaction, there can be mentioned alkaline phosphatase, peroxidase, β-galactosidase and so forth. The antigen peptide that can be detected by using an antibody is not particularly limited so long as it has antigenicity and its localization can be detected based on an antigenantibody reaction. Specifically, there can be mentioned a histidine polymer (His tag), a peptide sequence YPYDVPDYA (SEQ ID NO: 19, HA tag) contained in the hemagglutinin protein of influenza virus, a peptide of which N-terminus or C-terminus is bonded to either one of these and so forth.
Examples of the peptide that can specifically bind to another substance include avidin or streptavidin, which specifically binds to biotin, or protein A which specifically binds to an immunoglobulin, and so forth.
A method for constructing a gene library of genes coding for fusion proteins of a marker protein and a part or whole of a search object protein as described above will be explained below.
The gene library utilizes a vector DNA suitable to transiently express a fusion protein in a cell as a basic structure. As the vector, a plasmid vector or viral vector is usually used. Preferably, a so-called expression vector is used, which includes an expression regulatory sequence such as a promoter that enables expression in a eukaryotic cell.
A gene coding for a fusion protein is obtained by ligating DNA coding for a marker protein (marker protein gene) and DNA coding for a search object protein (object gene). The manner of fusion of the marker protein and the search object protein is not particularly limited, but it is usually preferable to position the marker protein on the N-terminal side and the search object protein on the C-terminal side. A termination codon is eliminated from the marker protein gene beforehand so that translation of the fusion protein should not be terminated. Further, DNA coding for a polypeptide such as a linker may be disposed between the marker protein gene and the object gene, but, in this case, an in-frame termination codon needs to be prevented from existing.
The fusion protein gene is inserted into a vector DNA in such a manner that a gene coding for the fusion protein can be expressed. Concrete procedures for constructing a fusion gene library will be exemplified below.
First, in order to prepare a cloning vector for cloning an object gene, a marker protein gene is inserted into a multi-cloning site located immediately downstream from a promoter in an expression vector. At this time, a cloning site for insertion of the object gene is provided downstream from the marker protein gene. Subsequently, the object gene is inserted into a cloning site of the cloning vector. As the object gene, cDNA is typically used. As the cDNA, double-stranded DNA is used, which is obtained from a single-stranded cDNA synthesized by reverse transcription using mRNA prepared from a cell or tissue as a template. The cDNA may include the whole or a part of a coding region. Since mRNA prepared from a cell or tissue is a mixture of those transcribed from many kinds of genes, a library including many kinds of genes is constructed by using such mRNA as a starting material for constructing the library. It is preferred that as many kinds as possible of cDNA are cloned in the library, but vector DNA and cDNA are preferably ligated so that one arbitrary kind of cDNA should be inserted into the cloning vector.
When a vector that does not include an expression regulatory sequence is used, an appropriate expression regulatory sequence is inserted upstream from the fusion protein gene.
The method of the present invention utilizes a phenomenon that, when a cell receives a stimulus such as stimulus by a drug, a transcription factor or cofactor in cytoplasm is specifically transported into a nucleus in response to the stimulus, and one of its objects is to clone a gene coding for such a transcription factor or cofactor. As means for achieving this object more efficiently, a cDNA library described below can be constructed.
A usual cDNA library includes many kinds of genes, and among them are genes coding for proteins that are transported into a nucleus without receiving any stimulus. As such proteins that are transported into a nucleus without receiving any stimulus, for example, structural proteins, which are components of the nucleus, can be mentioned. The structural proteins constituting a nucleus are synthesized in cytoplasm and then transported into the nucleus irrespective of presence or absence of a stimulus from the outside so as to function as components of the nucleus. There are many examples of the structural proteins constituting a nucleus, and these include proteins found in a structure called nuclear lamina considered to contain intermediate filaments supporting the inside of the nuclear membrane, histones and non-histone chromosomal proteins, which bind to DNA to play many roles, and so forth.
Accordingly, it becomes possible to efficiently obtain the object genes by constructing a library from which the aforementioned genes that may cause false positives in search of the object genes are eliminated beforehand. In the present invention, such a library from which genes that may cause false positives are eliminated is referred to as a cleaned library. It is preferred that unnecessary genes should be completely eliminated from the cleaned library, but even when the elimination is incomplete, the effect of the library can be sufficiently exhibited when most of such genes are eliminated.
An example of the method for constructing a cleaned library will be explained below. First, a library to be used as a base is constructed and introduced into cells to express fusion proteins therein. When GFP is used as a marker protein, localization of the fusion proteins can be detected by fluorescence of GFP. Further, the cells are fractioned by using a cell sorter without applying a stimulus such as stimulus by a drug. At this time, when genes coding for nucleus-constituting proteins or the like as described above are included, fusion proteins including such genes can be identified since these proteins are transported into nuclei even without a stimulus and the nuclei emit fluorescence. Since these cells of which nuclei emit fluorescence are not targeted, these are separated by using a cell sorter and removed. Since the remaining cell fraction does not include cells introduced with genes coding for proteins emitting fluorescence in a nucleus without a stimulus, these cells are collected, and plasmid DNAs are recovered from the fraction to construct a cleaned library. Thus, there are no or very few fusion proteins that are transported into the nucleus without a stimulus in the obtained cleaned library obtained as described above. Therefore, if the subsequent operations are performed by using this cleaned library, genes coding for proteins that are transported into nuclei in response to a stimulus such as stimulus by a drug can be efficiently obtained, and thus such a cleaned library is useful.
Further, many genes included in a cDNA library include those coding for proteins that are never transported into nuclei such as, for example, membrane proteins binding to cell membranes or proteins secreted out of the cells. Elimination of genes coding for such unnecessary proteins from the library beforehand is effective to improve efficiency in obtaining genes of proteins that are transported into nuclei in response to a stimulus such as stimulus by a drug, and such a library can also be included in the scope of the cleaned library.
An example of the method for constructing such a library will be explained below. There is a method for separating genes coding for proteins having a nuclear transport signal and genes coding for proteins not having the signal in an experiment system using yeast (Nature Biotechnology, 16, 1338–1342 (1998)). In this method, for example, genes having a nuclear transport signal such as a transcription factor NFκB and genes that do not have the signal can be separated. First, cDNA to be used as a base is prepared by reverse transcription of mRNA, cloned into vector described in the aforementioned reference and introduced into yeast to express proteins. Since this vector is designed so that auxotrophy should change and thus yeast can grow only when the expressed proteins are transported into nuclei, yeast colonies can be obtained only when the expressed proteins are transported into nuclei. When many of these colonies are collected and cloned cDNA are recovered from the colonies, there are no or very few proteins such as cell membrane proteins, which are not transported into the nuclei, among the encoded proteins. The cDNA thus obtained are cloned again into vectors GFP-F1, GFP-F2 and GFP-F3, which are described later, to construct a library. When a cleaned library constructed as described above is used to perform subsequent operations, genes coding for proteins that are transported into nuclei in response to a stimulus such as stimulus by a drug can be readily selected since genes that are not involved in the nuclear transport at all are eliminated. Thus, this cleaned library is useful.
As described above, there are various methods for constructing a cleaned library. Each of these may be solely employed, or a cleaned library may be first constructed by one method and then cleaning may be further performed repeatedly by other methods.
Further, since genome nucleotide sequences of various species including human have been elucidated, it has become possible to estimate genes coding for DNA binding proteins and transcription regulatory factors from genome nucleotide sequences by gene sequencing. For example, it has become possible to find genes coding for proteins having DNA-binding motifs such as zinc fingers among genome nucleotide sequences by bioinformatics. However, since computer analysis alone cannot show what functions these genes exhibit when expressed as proteins, it is necessary to express these proteins and perform experiments to analyze their functions. However, according to the present invention, the following experiment is enabled. First, a plurality of kinds of candidate genes that interest experimenters are listed based on bioinformatics or other information sources. The number of the candidate genes may be several dozens or several thousands, or even more. Then, based on information obtained from bioinformatics or the like, interesting genes can be collected by obtaining those provided by public institutions (for example, The Institute of Physical and Chemical Research (RIKEN), Tsukuba-shi) or commercially available cDNA. Then, a library of genes expressing GFP fusion proteins is constructed by using the collected candidate genes, and the present invention is carried out by using this library. Since the library is constructed by collecting candidate genes that interest the experimenters, nuclear transport proteins that interest the experimenters can be more efficiently detected. That is, since interesting genes are searched for in a small population that is limited beforehand, this method is efficient and useful as a method for carrying out the present invention.
Further, a nuclear membrane has a tunnel-like structure called a nuclear pore, which connects the inside of a nucleus and cytoplasm. Through the nuclear pore, substances are selectively transported into and out of the nucleus. Usually, a protein having a size of 40 kDa or larger cannot pass through the nuclear pore. However, when a protein has a special nuclear transport signal or is bound to a special protein that is responsible for transport, even a protein having a large size may be selectively transported into the nucleus. That is, it can be considered that passage through the nuclear pore is highly selectively regulated. On the other hand, small proteins may nonspecifically pass through the nuclear pore and flow into the nucleus.
In the examples of the present invention, GFP fusion proteins are used. GFP itself has a size of about 27 kDa. Therefore, if a protein fused with GFP is extremely small, the size of the whole fusion protein becomes not more than 40 kDa, and hence the fusion protein may flow into the nucleus nonspecifically. In this case, the nonspecific inflow increases false positives in gene search of the present invention.
Accordingly, in order to reduce false positives, the following procedure can be carried out. A nuclear export signal (Nature Biotechnology, 16, 1338–1342 (1998)) is incorporated into an amino acid sequence of a fusion protein to be expressed. Specifically, since the nuclear export signal is a short amino acid sequence, the incorporation can be carried out by adding a nucleotide sequence coding for the signal to a portion coding for the GFP fusion protein in the library. As a result, a GFP fusion protein having a nuclear export signal is expressed. Then, since this protein is actively discharged out of the nucleus by the nuclear export signal, a protein that nonspecifically flows into the nucleus due to diffusion can be eliminated out of the nucleus. As a result, false positives can be reduced.
When this method is used, true nuclear transport proteins are also pushed back out of the nucleus. However, since this is not the case where transport into the nucleus and transport out of the nucleus are balanced and no protein exists in the nucleus, fluorescence can be detected by a cell sorter and cells can be collected.
A gene library obtained as described above is introduced into appropriate eukaryotic cells. As the eukaryotic cells, animal cells, insect cells and plant cells can be mentioned. As the animal cells, there can be mentioned cell strains derived from tissues such as brain, muscles, placenta, pancreas and kidney of mammals such as human, monkey, mouse, rat and hamster. Specifically, there can be mentioned HepG2, which is an established cell line derived from human hepatoma (available from RIKEN Gene Bank Cell Bank, American Type Culture Collection (ATCC) or the like), COS-7, which is an established cell line derived from kidney of monkey (also available from RIKEN Gene Bank Cell Bank, American Type Culture Collection (ATCC) or the like) and so forth.
In order to introduce library DNA into aforementioned cells, methods usually used to introduce DNA into cultured cells such as the lipofection method, calcium phosphate method, DEAE dextran method, electroporation method, microinjection method, particle gun method and method using a virus vector can be used. Further, commercially available gene introducing reagents may also be used.
In the above-described operations, preparation of mRNA, preparation of cDNA by reverse transcription, ligation of a DNA fragment and a vector, introduction of a vector into a cell, expression of a gene and so forth can be performed according to general methods described in protocol collections such as Sambrook, J., Fritsch, E. F., and Maniatis, T., “Molecular Cloning A Laboratory Manual, Second Edition”, Cold Spring Harbor Laboratory Press, 1.21 (1989). Further, libraries similar to the gene library of the present invention are described in Journal of Cell Biology, 146, 29–43 (1999) and so forth.
Subsequently, fusion proteins are expressed in transgenic cells to produce the fusion proteins. When a constitutional promoter is used as a promoter for expressing the fusion protein genes, no special operation is required. However, when a inducible promoter is used, an operation required for induction is performed.
(2) Step (b)
Subsequently, the transgenic cells are divided into two groups, and only cells of one group are stimulated so that nuclear transport proteins should be transported into nuclei.
The nuclear transport proteins are stimulated for the nuclear transport by a chemical substance, physiologically active substance, environmental change, contact with other cells or the like. As the chemical substance, there can be mentioned nutrients such as amino acids and fatty acids, dioxin, alcohol and so forth. As the physiologically active substance, tumor necrosis factor α (TNFα), various interleukins, insulin, Fas Ligand and so forth. These substances include not only substances that have been reported for their stimulus for nuclear transport to date but also those expected to be discovered in future. Further, as the environmental change, there can be mentioned a thermal stimulus such as high or low temperature, pH change and so forth. As the “other cells”, there can be mentioned contact of a killer cell with a target cell and so forth.
In this step, among the transgenic cells divided into two groups, a stimulus as described above is applied to cells of one group, but not to those of the other group. Application of a stimulus or no stimulus is a relative expression, and a case where a strong stimulus is applied to cells of one group and a weak stimulus is applied to cells of the other group is also encompassed in the scope of the present invention.
(3) Step (c)
In this step, for each of the stimulated cell group and the unstimulated cell group, localization of the fusion proteins in cells is detected based on the marker protein, and cells in which localization of the fusion proteins is observed in the nuclei or their nuclei are isolated.
The fusion proteins can be appropriately detected depending on the kind or property of the marker protein. For example, when a protein emitting self-fluorescence is used as the marker protein, the fusion proteins can be detected by observing fluorescence. When a protein catalyzing a color development reaction is used, cells can be immersed in a solution containing a substrate matching the protein, for example, when alkaline phosphatase is used, BCIP or NBT, which is a substrate for color development, and an enzymatic reaction can be allowed to obtain color development of a site in which the marker protein is localized. When an antigen peptide is used, cells can be immunostained by using a labeled antibody obtained by labeling an antibody binding to the antigen with a fluorescent dye such as FITC, TRITC or DTAF or an enzyme, and can be detected. Similarly, when a peptide such as avidin is used, it can also be detected in the same manner as in the method using an antigen by labeling a ligand such as biotin, which binds to avidin, with a fluorescent dye or an enzyme. These methods are usual cell biological methods, and there are many established methods. Further, the present invention is not limited to these methods, and any method may be used so long as the marker protein can be detected.
A specific detection method utilizing an antigen peptide as the marker protein will be exemplified below. Transgenic cells expressing the fusion proteins are immobilized with methanol, acetone, formaldehyde or the like. At this time, in order to clarify whether staining of the nuclei of the cells expressing the fusion proteins is present or absent, it is effective to allow the fusion proteins in the cytoplasm to flow out of the cells by treating the cells with digitonin prior to immobilization so that permeability of the cell membranes should be increased. After the cells are immobilized, they are rinsed with a buffer such as PBS, and then treated with an anti-peptide antibody diluted to an appropriate concentration. The anti-peptide antibody binds to an antigen peptide portion of the fusion protein. If this antibody is directly labeled with a fluorescent dye, localization of the fusion proteins can be detected by using the fluorescence as a marker, and whether the fusion proteins exist in the nuclei can be examined. Further, when the anti-peptide antibody is used as a primary antibody, and an antibody recognizing and binding to the primary antibody is used as a secondary antibody, luminosity is increased by an amplification effect and thus sensitivity can be improved.
Subsequently, cells in which localization of the fusion proteins in the nuclei is observed or their nuclei are isolated. Even when the fusion proteins are localized in the nuclei, the fusion proteins may exist in cytoplasm. In such a case, in order to clarify the presence or absence of fluorescence, color development or the like of the fusion proteins in the nuclei and thereby make identification reliable, it is preferred that the nuclei are isolated by disrupting the cells in order to separate the nuclei in which localization of the fusion proteins is observed. Further, it is also effective to allow the fusion proteins in the cytoplasm to flow out of the cells by treating the cells with digitonin or the like to increase permeability of the cell membranes.
The nuclei can be isolated by, for example, the method shown below (refer to “Methods of Cultured Cell Experiment for Molecular Biology Study”, Yodosha, pp. 141–143). Cells are suspended in a buffer cooled with ice and disrupted on ice by using a homogenizer. The cell-disrupted suspension is overlaid on a buffer having an appropriate density and centrifuged so that only a nuclear fraction should be recovered as precipitates.
Cells in which fluorescence is detected in the nuclei or their nuclei can be isolated by cell sorting, flow cytometry, microdissection, microscopic observation or the like. Cell sorting is a method wherein a suspension of cells or their nuclei is made a thin flow so that the cells or their nuclei should flow in one line, and each cell or nucleus is successively irradiated with laser beams to detect fluorescence. When desired fluorescence is detected, an electric field is instantaneously applied to the cell or nucleus to blow the cell or nucleus horizontally so as to be collected in a tube. In the present invention, nuclei in which fluorescence is detected or cells containing such nuclei are collected. Cell sorting is an established technique, and a cell sorter, which is an apparatus used therefor, is commercially available.
Further, as another method, there can be mentioned a method of observing cells with a microscope, identifying desired cells and collecting the cells. An example of the method comprising collecting desired cells from many cells on a culture plate or slide glass will be explained below. Cells on a culture dish are collected by subjecting the cells that adhere to the dish to trypsin treatment and removing the cells. In order to minimize contamination of cells that are not the target cells at this time, trypsin can be injected into a plastic thin tube while a ring at an end of the tube is placed so that the target cells should be included, and the target cells and only a small number of cells around them can be removed from the dish and collected. Since cells are small, it is difficult to completely prevent contamination of surrounding cells that are not the target cells even when a thin tube is used, but isolation accuracy can be improved by repeating this isolating operation.
Further, excellent methods for collecting individual cells on a slide glass have already been developed. Similar systems are commercially available from a plurality of microscope manufacturers, but LM 200 Microdissection System available from Olympus Optical Co., Ltd. is most commonly used. This system is composed of an inverted microscope and an accompanying cell-collecting device. First, cells are observed with the microscope to identify the cells whose nuclei emit fluorescence. Then, the cells are irradiated with laser beams. A block of the cell-collecting device is brought into contact with cells from above, and the contact surface of the block with the cells is melted by heat of the laser and strongly adheres to cells. Since the light flux of the laser is narrowed to the size of cells, only the target cells can adhere to the block without contamination of surrounding cells. When a plurality of cells are to be collected, operations of observation, cell selection and laser irradiation are repeated. Then, when the block is finally lifted up, only cells desired to be collected adhere to the surface of the block and are lifted up together with the block. Thus, only the targeted cells can be collected.
Further, as another method, there is also a method of introducing a cell suspension into each well of a 96-well plate or 384-well plate so that a small number of cells should be added to each well, distinguishing cells showing desirable protein localization by microscopic observation, and collecting the cells from each well.
Further, any other methods can be used so long as localization of fusion proteins can be detected to isolate cells or their nuclei.
(4) Step (d)
In this step, genes coding for fusion proteins are recovered from isolated cells or their nuclei, and genes recovered from cells isolated from the stimulated cell group and genes recovered from cells isolated from the unstimulated cell group are compared. Genes can be recovered in the same manner as in usual recovery of plasmids from cells or their nuclei (refer to “Bio-Manual Series 3, Experimental Procedures for Gene Cloning”, Yodosha, p.114–115). The genes can be compared by sequence analysis of a full-length or a part of a portion coding for a search object protein. Sequence analysis may be performed for the full length of a cloned fragment or only a partial region, for example, a fragment excised with a restriction enzyme.
As a result of comparison of the genes, a gene coding for a nuclear transport protein can be obtained by identifying a gene recovered only from the stimulated cell group or a gene recovered only from the unstimulated cell group.
A gene group obtained from the unstimulated cell group is considered to include genes coding for nucleoproteins constituting the nuclei and so forth. These proteins are fractioned as those transported into the nuclei since they are transported into the nuclei irrespective of presence or absence of a stimulus. On the other hand, the genes obtained from the stimulated cell group include genes coding for proteins that are transported into the nuclei in response to a stimulus, but also includes the aforementioned genes obtained from the unstimulated cell group. Thus, a gene coding for a protein which is transported into the nucleus specifically in response to a stimulus can be identified by comparing the genes obtained from these two cell groups.
Typically, sequence analysis is performed for all the obtained genes to list all the genes recovered from both the cell groups, and a gene found only in the stimulated cell group is identified. Since the gene found in this procedure is a gene coding for a protein which is transported into the nucleus only when a stimulus is applied, it is a gene coding for a transcription factor, cofactor or the like and considered to be an important gene. Thus, an important gene can be found.
As described above, a gene coding for a nuclear transport protein can be isolated or concentrated from the cDNA library. Further, in step (c), concentration of the object gene can be increased by repeating, for the isolated cells, the operations of detecting localization of fusion proteins in the cells utilizing the marker protein and isolating cells in which localization of the fusion proteins is observed in the nuclei or their nuclei.
<2> Utilization of the Present Invention
For example, there is an important cytokine protein called interferon γ, which stimulates a cell. This protein is present in blood, binds to a receptor on the outer surface of a cell membrane to transmit a signal into a cell and thereby induces various phenomena such as anti-viral activity of the cell. STAT1 protein is known as one of transcription factors for transmitting a signal of interferon γ from the receptor into the nucleus. It is known that, when a stimulus of interferon γ is applied, STAT1 in the cytoplasm is transported into the nucleus, binds to DNA and induces various gene expressions. As a result, anti-viral activity is obtained.
By utilizing the method of the present invention, transcription factors such as this STAT1 can be identified or obtained. Further, if an unknown transcription factor is transported into the nucleus in addition to STAT1 by the stimulus of interferon γ, it can also be found.
Thus, genes coding for proteins transmitting a signal into nuclei in response to various stimulus from the outside, which are important for life, can be identified by the present invention. These may include genes whose sequences have been known but whose functions are unknown, and novel transcription factors or cofactors that have been unknown to date can also be found.
Detection of the transcription factors or cofactors described above can be utilized for, for example, screening for development of drugs (anti-viral agents) for enhancing anti-viral activity. For example, if a novel transcription factor responsible for anti-viral activity is found by the present invention, a small molecule compound having an action for transporting the transcription factor into the nucleus can then be screened. Thus, there is given a possibility of developing drugs that enhance anti-viral activity by transporting the transcription factor into the nuclei.
Further, if, for example, a transcription factor involved in amelioration of diabetes mellitus is found, an antidiabetic drug can be developed by screening a drug that activates the transcription factor or transports it into the nuclei.
Further, if a transcription factor such as NFκB involved in Inflammatory reaction is found, a drug inhibiting Inflammation can be developed by screening a drug that blocks the nuclear transport so that the transcription factor should not be transported into the nuclei.
Further, if a transcription factor enhancing salt damage resistance or cold resistance of a plant is found, cold resistance or salt damage resistance can be enhanced by finding a method for transporting the transcription factor into the nuclei.
As described above, it is considered that, if a novel transcription factor or a novel function of a known transcription factor is found, it leads to screening of a novel drug or pharmaceutical agent, development of a genetically recombinant plant having improved characteristics by gene modification or the like, and so forth.
<3> Method for Screening Therapeutic Agent for Disease Caused by TNF Stimulus
The second aspect of the present invention is a method for screening a therapeutic agent for a disease caused by TNF stimulus and comprises the following steps of:    (a) preparing eukaryotic cells expressing a protein which is transported from cytoplasm into a nucleus through a nuclear pore in response to TNF stimulus;    (b) allowing TNF and a test substance to act on the eukaryotic cells;    (c) detecting a protein which is transported into the nucleus; and    (d) selecting a test substance that accelerates or inhibits the nuclear transport of the protein when the test substance is allowed to act on the cells in comparison to a case where the test substance is not allowed to act on the cells.
Hereafter, the aforementioned method will be explained step by step.
(1) Step (a)
First, eukaryotic cells expressing a protein which is transported from cytoplasm into nuclei through a nuclear pore in response to TNF stimulus are prepared. Such eukaryotic cells are, for example, cells introduced with a gene coding for a nuclear transport protein so that the protein should be expressed. These are typically cells harboring a gene identified as a gene coding for a nuclear transport protein by stimulating the nuclear transport protein so that it should be transported into a nucleus using TNF in the aforementioned method according to the first aspect of the present invention. If a fusion protein with a marker protein whose localization in a cell can be detected is used as the nuclear transport protein, the nuclear transport of the nuclear transport protein can be detected by using the marker. As the marker protein and the method for detecting the marker, the same ones as in the first aspect of the invention can be used. Further, specific examples of the nuclear transport protein include the proteins shown in Table 9 described later, but they are not limited to them. Gene products that can be selected by the first aspect of the invention can be preferably used.
(2) Step (b)
Subsequently, TNF and a test substance are allowed to act on the eukaryotic cells. In order to allow the test substance to act on the eukaryotic cells, for example, cells can be immersed in a solution of a test substance so that the substance should be taken into the cells from the outside of the cells, or the eukaryotic cells can be transformed with a foreign gene that is not a gene coding for the nuclear transport protein so that the gene should be expressed. In the latter method, the expression product of the foreign gene serves as the test substance. As such eukaryotic cells, transformants transformed with a gene library can be mentioned.
In this step, eukaryotic cells on which the test substance is not allowed to act, but only TNF is allowed to act are prepared as a control. If the gene library is constructed by using a vector including a-promoter capable of regulating expression, presence or absence of the test substance can be controlled. The library can be constructed in the same manner as in the first aspect of the invention.
(3) Step (c)
In the eukaryotic cells on which TNF is allowed to act in the step (b), localization of the nuclear transport protein in the cells is detected. This operation can be performed in the same manner as in the detection of localization of the fusion proteins in the cells in the first aspect of the invention.
(4) Step (d)
Subsequently, there is selected a test substance that accelerates or inhibits the nuclear transport of the nuclear transport protein when the test substance is allowed to act on the cells in comparison to the case where the test substance is not allowed to act the cells.
The substance selected in this step is a substance that specifically inhibits or accelerates the nuclear transport of the nuclear transport protein or a substance that inhibits or accelerates production of the nuclear transport protein itself.
Further, a substance that accelerates or inhibits the nuclear transport of the nuclear transport protein can be obtained based on the gene sequence of the nuclear transport protein. If a known nuclear transport signal sequence is found in the gene sequence, a substance binding to the sequence is designed. Further, if a known nuclear transport signal sequence is not found in the gene sequence, the gene sequence is partially deleted to identify a site for inhibiting nuclear transport, and a substance binding to this site is designed. As such a substance, for example, anti-sense DNA or ribozyme can be mentioned. Further, a signal specific for TNF can also be enhanced by introducing a gene coding for a nuclear transport protein into the cells. Furthermore, a protein that binds to a nuclear transport protein can be obtained by the yeast two-hybrid method or the like, and a substance inhibiting binding of the two can be screened or designed.
By administering a substance obtained by the method of the present invention together with TNF to a liver cell strain, primary culture liver cell or animal, for what diseases the substance should be developed as a therapeutic agent can be known depending on what actions of TNF are inhibited or accelerated. As the TNF actions, release of cytokine, cell growth, cell differentiation, apoptosis induction, gene expression induction and so forth can be mentioned depending on the target cells. Release of cytokine can be detected by measuring the cytokine by the ELISA method. Further, cell growth can be determined based on, for example, uptake of isotope-labeled thymidine. Cell differentiation can be determined by, for example, staining a differentiation marker with an antibody and measuring it by FACS. Apoptosis induction can be determined by, for example, the tunnel method. Induction of gene expression can be determined by, for example, using a gene chip or PCR.
Specific examples of the targeted diseases of the method of the present invention include liver diseases such as acute hepatitis, chronic hepatitis, fulminant hepatitis, liver fibrosis, cirrhosis and liver cancer, and there can be further mentioned Crohn's disease, multiple secrosis, chronic rheumatoid arthritis, systemic vasculitis, septic shock, diabetes mellitus (insulin resistant) and so forth.
By combining the first and second aspects of the invention, a gene coding for a nuclear transport protein can be retrieved, and a therapeutic agent for a disease caused by TNF stimulus can be screened by using the identified gene.